Ad5-spike COVID-19 vaccine does not aggravate heart damage after ischemic injury


 Hopes for a COVID-19 vaccine are now a reality. The spike protein of SARS-CoV-2, which majorly binds to the host receptor ACE2 for cell entry, is used by most of the COVID-19 vaccine candidates as a choice of antigen. ACE2 is highly expressed in the heart and is known to be protective in multiple organs. Interaction of spike with ACE2 has been reported to reduce ACE2 expression and affect ACE2-mediated signal transduction in the heart. However, whether a spike-encoding vaccine will aggravate myocardial damage after a heart attack via affecting ACE2 remains unclear. Therefore, for patients with or at risk of heart diseases, questions arise around the safety of the spike-based vaccines. Here, we demonstrate that ACE2 is up-regulated and protective in the injured mouse heart after myocardial ischemia/reperfusion (I/R). Infecting human cardiomyocyte, smooth muscle cells, endothelial cells, and cardiac fibroblasts with a recombinant adenovirus type-5 vectored COVID-19 vaccine expressing the spike protein (AdSpike) does not affect cell survival and cardiomyocyte function, whether the cells are subjected to hypoxia-reoxygenation injury or not. This observation is further confirmed in human engineered heart tissues. Furthermore, AdSpike vaccination does not aggravate heart damage in wild-type or humanized ACE2 mice after I/R injury, even at a dose that is ten-fold higher as used in human. This study represents the first systematic evaluation of the safety of a leading COVID-19 vaccine under a disease context and may provide important information to ensure maximal protection from COVID-19 in patients with or at risk of heart diseases.

vaccine expressing the spike protein (AdSpike) does not affect cell survival and cardiomyocyte function, whether the cells are subjected to hypoxia-reoxygenation injury or not. This observation is further confirmed in human engineered heart tissues. Furthermore, AdSpike vaccination does not aggravate heart damage in wild-type or humanized ACE2 mice after I/R injury, even at a dose that is ten-fold higher as used in human. This study represents the first systematic evaluation of the safety of a leading COVID-19 vaccine under a disease context and may provide important information to ensure maximal protection from COVID-19 in patients with or at risk of heart diseases. Gu et al. / 3 The ongoing pandemic of coronavirus disease 19 (COVID-19) caused by a coronavirus SARS-CoV-2 has spurred an unprecedented public health crisis worldwide. Hence, the development of a safe and effective vaccine that can prevent SARS-CoV-2 infection and transmission has rapidly become top priority. For cell entry, the SARS-CoV-2 virus majorly binds to the host receptor angiotensin-converting enzyme 2 (ACE2) through its spike glycoprotein, which is the only viral protein that interacts with host cells and is the most diverging protein between different coronaviruses [1]. Therefore, generating a vaccine encoding/introducing the spike protein is the strategy used by the majority of COVID-19 vaccine candidates, including vaccines based on viral vectors, nanoparticles/virus-like particles, proteins/peptides, RNA, and DNA [2]. There has been an unprecedented rapid response by vaccine developers with now over 60 COVID-19 vaccine candidates in clinical trials and ten having been approved for at least limited use as of 1 March 2021. Although trials of the approved spike-based COVID-19 vaccines have not detected vaccinerelated serious adverse events, it should be noticed that safety of these vaccines were mostly evaluated in healthy volunteers, and little is known about their effects on patients with or at risk of chronic diseases [3][4][5].
ACE2 is a membrane-localized aminopeptidase that is highly expressed in the heart and blood vessels and has direct effects on cardiac function and multiple organs via counter-regulation of the renin-angiotensin system (RAS), a primary cardiovascular regulatory system [6]. Increased ACE2 expression is observed after myocardial infarction in both rodent and human [7], suggesting that ACE2 may also be involved in regulating heart repair following ischemic injury. We have confirmed the significant increase of ACE2 in the injured mouse heart after myocardial ischemia/reperfusion (I/R) (Fig. 1A-B). Furthermore, we found that ACE2 overexpression reduced the infarct size and improved heart function during I/R, whereas ACE2 knockdown aggravated heart damage ( Fig. 1C-F). These data suggest that ACE2 has a protective role during myocardial I/R. However, interaction of the spike protein with ACE2 during virus infection has been shown to reduce ACE2 expression [8,9] and alter the RAS signal transduction. For example, binding of spike to ACE2 upregulates the Ras-ERK-AP-1 pathway leading to activation of pro-remodeling factors such as the C-C motif chemokine ligand 2 (CCL2), which may contribute to cardiac injury and subsequently cause the fibrosis associated with disease manifestation [10].This raises a pivotal question: will the vaccine encoding the spike protein increase the risk of myocardial damage after a heart attack via binding to and affecting the cardiac ACE2? For patients with coronary or ischemic heart disease or people at high heart attack risk (e.g., arrhythmias, hypertension, or diabetes mellitus), questions arise around the safety after COVID-19 vaccinations in the setting of increased ACE2 expression in the injured heart and warrant close investigation. Thus, in this study, we evaluate the effects of spike-expressing vaccine on hearts that subject to I/R injury using human cardiac cells, engineered human heart tissues, and humanized ACE2 mice ( Supplementary Fig.   S1).
We firstly examined the ex vivo effect of the spike-based vaccine on the four major cell types of human heart ( Supplementary Fig. S2A), using cardiomyocytes (hCMs) and smooth muscle cells  Fig. 2A-B). Similar observations were also seen in rat cardiomyocytes of neonatal (nrCMs) or adult (arCMs) origins ( Supplementary Fig. S4). These data suggest that spikebased vaccine has little effect on the survival of cultured cardiovascular cells even after hyp-reox injury.
To explore whether AdSpike affects hCM function in both normoxia and hyp-reox situations, we assessed spontaneous intracellular Ca 2+ fluctuations in hCMs that infected by AdVector or AdSpike at various MOI using confocal laser microscopy. Individual hCM in each condition exhibited independent spontaneous beating and rhythmic Ca 2+ transients. We found that the amplitude, time to peak, time of decay to 63% peak (T-63% decay) of the Ca 2+ transients were similar in the AdVector and AdSpike groups, whether they were subjected to hyp-reox injury or not ( Fig. 2C; Supplementary Fig. S5A). Once again, we independently confirmed this conclusion in nrCMs ( Supplementary Fig. S5B). To further assess the calcium handling properties, we subjected AdVector-or AdSpike-infected hCMs, nrCMs, and arCMs to a series of increasing electrical field stimulation frequencies. We found that AdSpike group could keep pace with the To provide a further assessment of AdSpike on human heart tissue with or without ischemic injury, we took advantage of the human engineered heart tissue (hEHT) model ( Fig. 3A;   Supplementary Fig. S8) which widely expressed ACE2 (Fig. 3B). We infected the cells with AdSpike while fabricating the hEHTs (Fig. 3C; Supplementary Fig. S8A). The infection efficiency was very high as indicated by staining of the spike-fused flag epitopes (Fig. 3C), and no increased cell death was observed (shown by PI staining) ( Fig. 3D; Supplementary Fig. S9A). As unhealthy tissues always show abnormal morphology during hEHT culturing [11], we thus monitored the morphology of hEHTs by examining their width. The width of hEHTs decreased along with culture time, indicating that the hEHTs were gradually getting matured (Supplementary Fig. S9B). We traced more than 103 hEHTs for up to 10 days and compared the AdSpike-infected hEHTs with the AdVector control. Under both normoxia and hyp-reox conditions, we found that they exhibited similar width (Supplementary Fig. S9C) and spontaneous contraction properties, including amplitude and 50% peak time, albeit there was a slight increase in beating rate in hEHTs infected by high MOI AdSpike ( Fig. 3E; Supplementary Fig. S9D). Consistently, there was no difference between the AdVector and AdSpike groups in beating amplitude or 50% beating peak time under 1.5Hz electrical pacing ( Fig. 3F; Supplementary Fig. S9E). More importantly, we used a customized contractility force test system to analyze the hEHT's contraction in stepped raising stretching length (stretching ratio 0%, 2%, 4%, 6%) [11] (Supplementary Fig. S8D). We observed that the contractility force increased while stretching in both AdVector and AdSpike hEHT groups, whereas there was no significant difference in max contractility force and max/min contractility force ratio between the two groups ( Fig. 3G; Supplementary Fig. S9F). Similar results in contraction raising time were observed ( Fig. 3G; Supplementary Fig. S9F). In aggregate, the spikeencoding vaccine does not affect hEHT function in both normoxia and hyp-reox situations.
To further explore the in vivo effects of spike-based vaccine on hearts after myocardial I/R injury, we intramuscular injected AdSpike to wild-type C57BL/6 mice at a dose that is equal (1×10 9 viral particles per kilogram of body weight, AdSpike-low) or ten-fold higher (1×10 10 viral particles per kilogram of body weight, AdSpike-high) as used in human [12], subjected the mice to myocardial I/R injury 7 days post-vaccination, and examined the effects of AdSpike on heart function over a period of 28 days (Supplementary Fig. S10A). AdVector at a dose of 1×10 10 viral particles per kilogram of body weight was used as a control. I/R injury was created by temporal ligating the left anterior descending coronary artery in vivo for 1 hour followed by reperfusion.
Effective immunogenicity of AdSpike at both high or low dose was confirmed by the specific Gu et al. / 7 ELISA antibody responses to the receptor binding domain (RBD) 28 days post-vaccination (Fig.   4A). By high-resolution echocardiography, we found that heart function was equally preserved after AdSpike vaccination compared to the AdVector control, reflected by the left ventricular ejection fraction and fractional shortening (Supplementary Fig. S10B). Consistently, we observed comparable scar sizes and heart weight/body weights between the vaccinated and control mice ( Supplementary Fig. S10C-D). These data suggest that spike-based vaccine does not aggravate heart damage in wild-type mice after myocardial I/R injury.
It is known that the orthologous Ace2 receptor in mice has a lower affinity to bind the spike protein, therefore typical inbred mouse strains do not support robust SARS-CoV-2 infection and replication [13]. To exclude the possibility that the above observation was an experimental artifact caused by the inefficient binding between spike and the mouse Ace2, we utilized a humanized mouse model in which the human ACE2 coding-sequence was knock-in into the mouse Ace2 genomic locus and replaced its mouse ortholog ( Supplementary Fig. S10E-F). By using this model, we further confirmed that ACE2 expression was not significantly declined by AdSpike vaccination at both doses, whether subjected to myocardial I/R injury or not (Fig. 4B). In accordance with the observation in wild-type mice, histological and functional influences of AdSpike vaccination on I/R hearts of humanized ACE2 mice were neglectable when compared with the controls (Fig. 4C-E). Furthermore, neither the number of TUNEL + apoptotic cardiomyocytes or vascular density in the border zone was altered by AdSpike vaccination (Fig. 4F). Taken together, spike-based vaccine has no effect on cardiomyocyte apoptosis, scar formation, infarct revascularization, and function recovery during myocardial I/R injury.
Hopes for a COVID-19 vaccine are now a reality. Although the dominant pathology of COVID-19 involves the respiratory system, 20%-30% of COVID-19 patients experience severe cardiovascular damage, which emerged as a major indicator of poor prognosis [8,14]. Moreover, patients with pre-existing heart complications are more likely to develop severe illness and have higher risk of death compared with patients without co-morbidities [14,15]. Therefore, a COVID-19 vaccine would be lifesaving for patients with or at risk of heart diseases and warrants expedited Gu et al. / 8 vaccination. However, this group of people has been excluded from COVID-19 vaccine trials thus far and little is known about the performance of COVID-19 vaccines in them. Here, by combining models of human pluripotent stem cell-derived cardiac cells/tissues and humanized mouse models, we provide a proof-of-principle demonstration that spike-based Ad5 vaccine does not increase myocardial damage after ischemic injury. To our knowledge, this study represents the first systematic evaluation of the safety of a leading COVID-19 vaccine under a heart disease context.
Although there are still many unanswered questions, we expect the spike-based COVID-19 vaccine to be safe and at least partly effective after myocardial ischemic injury, and benefits of vaccination likely outweigh risks of vaccine-related adverse events. This study may not only pave the way for clinical trials of people with cardiovascular conditions receiving these vaccines, but also provide an example that may be adapted for elucidate the safety and efficacy of COVID-19 vaccines in other complex diseases context.   AdSpike has little effects on hEHTs that underwent spontaneous contraction, electrical eld stimulation, and mechanical tensile test in hyp-reox condition. (A) Low magni cation image of hEHT on day 21 (upper) and the equipment image of mechanical contractility force test for hEHT (lower). Scale bar, 2 mm.